Notes

  • Unclassified reads were excluded
  • Taxa with total reads number of 10 or lower were excluded
  • Eukaryotes were excluded from the analysis
  • Correlation statistic between ZMB and ZHMW : Pearson’s R^2
  • Only the top 25 taxa were used to calculate correlations for each level
  • Significantly Different Taxa Identification between ZMB and ZHMW : MAASLIN2
  • Statistic to find contribution of factors to the total variance: PERMANOVA
  • ALL taxa, NOT just the top 25 were used to calculate the PERMANOVA for each level
  • PERMANOVA model: Sex and Date as Fixed Effects and Account for Repeated Measures (Full model)

Import Exp data

Filter Exp data

Filter samples based on total read count

Fix sample names

Add lineage and metadata to Gold Standard Data

Limosilactobacillus fermentum in the theoretical composition was changed to Lactobacillus fermentum

## Error in validObject(.Object) : invalid class "otu_table" object: 
##  OTU abundance data must have non-zero dimensions.
## Error in dimnames(x) <- dn : 
##   length of 'dimnames' [1] not equal to array extent

Fix sample names from ‘-’

Merge exp data with GS

Filter Taxa for Gold Standard

These are the taxa that were found in the experimental data

##                         species taxid superkingdom         phylum
## 1             Bacillus subtilis  1423     Bacteria     Firmicutes
## 2         Enterococcus faecalis  1351     Bacteria     Firmicutes
## 3              Escherichia coli   562     Bacteria Proteobacteria
## 4 Limosilactobacillus fermentum  1613     Bacteria     Firmicutes
## 5        Listeria monocytogenes  1639     Bacteria     Firmicutes
## 6        Pseudomonas aeruginosa   287     Bacteria Proteobacteria
## 7           Salmonella enterica 28901     Bacteria Proteobacteria
## 8         Staphylococcus aureus  1280     Bacteria     Firmicutes
##                 class            order             family               genus
## 1             Bacilli       Bacillales        Bacillaceae            Bacillus
## 2             Bacilli  Lactobacillales    Enterococcaceae        Enterococcus
## 3 Gammaproteobacteria Enterobacterales Enterobacteriaceae         Escherichia
## 4             Bacilli  Lactobacillales   Lactobacillaceae Limosilactobacillus
## 5             Bacilli       Bacillales       Listeriaceae            Listeria
## 6 Gammaproteobacteria  Pseudomonadales   Pseudomonadaceae         Pseudomonas
## 7 Gammaproteobacteria Enterobacterales Enterobacteriaceae          Salmonella
## 8             Bacilli       Bacillales  Staphylococcaceae      Staphylococcus
##     count Gram Stain    EXP_sum
## 1 1740000          +         NA
## 2  990000          +   73.20904
## 3 1010000          - 6077.60892
## 4 1840000          +         NA
## 5 1410000          +         NA
## 6  420000          -         NA
## 7 1040000          -         NA
## 8 1550000          +         NA

Add Gram Stain Info

Omit Eukaryotes

Relative abundance of taxa at each taxonomic level

Gram Stain level

Phylum level

Class level

Order level

Family level

Genus level

Species level

Figure XB – no eval

B-diversity Analysis using sample-wise Bray-Curtis distances

Non-metric Multi-Dimensional Scaling (NMDS)

## Square root transformation
## Wisconsin double standardization
## Run 0 stress 0.1470839 
## Run 1 stress 0.1646013 
## Run 2 stress 0.1718542 
## Run 3 stress 0.1825241 
## Run 4 stress 0.1601833 
## Run 5 stress 0.1721381 
## Run 6 stress 0.1722551 
## Run 7 stress 0.1841597 
## Run 8 stress 0.1590276 
## Run 9 stress 0.1616144 
## Run 10 stress 0.181801 
## Run 11 stress 0.1674317 
## Run 12 stress 0.1669914 
## Run 13 stress 0.1750451 
## Run 14 stress 0.1505306 
## Run 15 stress 0.1615029 
## Run 16 stress 0.1744525 
## Run 17 stress 0.1736455 
## Run 18 stress 0.1548971 
## Run 19 stress 0.1766343 
## Run 20 stress 0.1763632 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##     20: stress ratio > sratmax

NMDS v1: color = DNA_isolation_method; shape = Vregion

NMDS v2: color = Vregion; shape = DNA_isolation_method

Statistics: Pearson’s Correlations, PERMANOVA

Parameters and Settings

Correlation

PERMANOVA model

Phylum level

Pearson’s between ZHMW or ZMB for each sample

Pairwise comparison of Taxon Abundance values between ZHMW and ZMB

##              taxon      p_value significant  label
##             <char>        <num>      <lgcl> <char>
## 1:  Actinobacteria 1.117457e-01       FALSE       
## 2:   Bacteroidetes 1.744864e-07        TRUE      *
## 3:   Cyanobacteria 3.234749e-01       FALSE       
## 4: Deferribacteres 2.941912e-01       FALSE       
## 5:      Firmicutes 8.161593e-10        TRUE      *
## 6:    Fusobacteria 2.203607e-07        TRUE      *
## 7:  Proteobacteria 8.974304e-05        TRUE      *
## 8:   Synergistetes 3.234749e-01       FALSE       
## 9:     Tenericutes 1.157477e-01       FALSE

PERMANOVA

Full-model PERMANOVA: source + dna.isolation.method

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Blocks:  strata 
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = otutab_t ~ sex + dna.isolation.methods + date, data = sampdat, method = distance, by = "terms", strata = sampdat$source)
##                       Df SumOfSqs      R2       F Pr(>F)    
## sex                    1   0.0586 0.00962  1.0000  0.001 ***
## dna.isolation.methods  1   1.1305 0.18547 19.2805  0.001 ***
## date                  13   1.1537 0.18927  1.5135  0.051 .  
## Residual              64   3.7526 0.61564                   
## Total                 79   6.0954 1.00000                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Order level

Pearson’s between ZHMW or ZMB for each sample

Pairwise comparison of Taxon Abundance values between ZHMW and ZMB

##                     taxon      p_value significant  label
##                    <char>        <num>      <lgcl> <char>
##  1:     Acidaminococcales 6.797769e-06        TRUE      *
##  2:         Aeromonadales 3.016794e-03        TRUE      *
##  3:     Anaeroplasmatales 1.157477e-01       FALSE       
##  4:            Bacillales 2.029339e-01       FALSE       
##  5:         Bacteroidales 1.746399e-07        TRUE      *
##  6:     Bifidobacteriales 1.673634e-01       FALSE       
##  7:       Burkholderiales 1.135086e-05        TRUE      *
##  8:     Campylobacterales 5.593058e-04        TRUE      *
##  9: Chroococcidiopsidales 3.234749e-01       FALSE       
## 10:         Clostridiales 1.765966e-13        TRUE      *
## 11:      Coriobacteriales 4.849193e-02        TRUE      *
## 12:     Deferribacterales 2.941912e-01       FALSE       
## 13:        Eggerthellales 3.811809e-01       FALSE       
## 14:      Enterobacterales 9.308357e-02       FALSE       
## 15:    Erysipelotrichales 1.672772e-01       FALSE       
## 16:       Fusobacteriales 2.203607e-07        TRUE      *
## 17:       Lactobacillales 9.958588e-03        TRUE      *
## 18:         Micrococcales 1.914214e-01       FALSE       
## 19:            Nostocales 3.234749e-01       FALSE       
## 20:       Oscillatoriales 3.234749e-01       FALSE       
## 21:        Pleurocapsales 3.234749e-01       FALSE       
## 22:       Pseudomonadales 2.368003e-01       FALSE       
## 23:       Selenomonadales 3.791984e-07        TRUE      *
## 24:       Synechococcales 3.234749e-01       FALSE       
## 25:        Tissierellales 3.234749e-01       FALSE       
##                     taxon      p_value significant  label

PERMANOVA

Full-model PERMANOVA: source + dna.isolation.method

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Blocks:  strata 
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = otutab_t ~ sex + dna.isolation.methods + date, data = sampdat, method = distance, by = "terms", strata = sampdat$source)
##                       Df SumOfSqs      R2       F Pr(>F)    
## sex                    1   0.2456 0.02440  2.8907  0.001 ***
## dna.isolation.methods  1   1.9654 0.19524 23.1302  0.001 ***
## date                  13   2.4172 0.24012  2.1882  0.001 ***
## Residual              64   5.4383 0.54023                   
## Total                 79  10.0666 1.00000                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Genus level

Pearson’s between ZHMW or ZMB for each sample

Pairwise comparison of Taxon Abundance values between ZHMW and ZMB

##                     taxon      p_value significant  label
##                    <char>        <num>      <lgcl> <char>
##  1:     Acidaminococcales 6.797769e-06        TRUE      *
##  2:         Aeromonadales 3.016794e-03        TRUE      *
##  3:     Anaeroplasmatales 1.157477e-01       FALSE       
##  4:            Bacillales 2.029339e-01       FALSE       
##  5:         Bacteroidales 1.746399e-07        TRUE      *
##  6:     Bifidobacteriales 1.673634e-01       FALSE       
##  7:       Burkholderiales 1.135086e-05        TRUE      *
##  8:     Campylobacterales 5.593058e-04        TRUE      *
##  9: Chroococcidiopsidales 3.234749e-01       FALSE       
## 10:         Clostridiales 1.765966e-13        TRUE      *
## 11:      Coriobacteriales 4.849193e-02        TRUE      *
## 12:     Deferribacterales 2.941912e-01       FALSE       
## 13:        Eggerthellales 3.811809e-01       FALSE       
## 14:      Enterobacterales 9.308357e-02       FALSE       
## 15:    Erysipelotrichales 1.672772e-01       FALSE       
## 16:       Fusobacteriales 2.203607e-07        TRUE      *
## 17:       Lactobacillales 9.958588e-03        TRUE      *
## 18:         Micrococcales 1.914214e-01       FALSE       
## 19:            Nostocales 3.234749e-01       FALSE       
## 20:       Oscillatoriales 3.234749e-01       FALSE       
## 21:        Pleurocapsales 3.234749e-01       FALSE       
## 22:       Pseudomonadales 2.368003e-01       FALSE       
## 23:       Selenomonadales 3.791984e-07        TRUE      *
## 24:       Synechococcales 3.234749e-01       FALSE       
## 25:        Tissierellales 3.234749e-01       FALSE       
##                     taxon      p_value significant  label

PERMANOVA

Full-model PERMANOVA: source + dna.isolation.method

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Blocks:  strata 
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = otutab_t ~ sex + dna.isolation.methods + date, data = sampdat, method = distance, by = "terms", strata = sampdat$source)
##                       Df SumOfSqs      R2       F Pr(>F)    
## sex                    1   0.3503 0.02341  2.8762  0.001 ***
## dna.isolation.methods  1   3.2119 0.21461 26.3718  0.001 ***
## date                  13   3.6093 0.24116  2.2796  0.001 ***
## Residual              64   7.7948 0.52082                   
## Total                 79  14.9663 1.00000                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Species level

Pearson’s between ZHMW or ZMB for each sample

Pairwise comparison of Taxon Abundance values between ZHMW and ZMB

##                                     taxon      p_value significant  label
##                                    <char>        <num>      <lgcl> <char>
##  1: Anaerobiospirillum succiniciproducens 4.820577e-03        TRUE      *
##  2:                    Blautia glucerasea 1.705144e-11        TRUE      *
##  3:                      Blautia hansenii 3.010974e-01       FALSE       
##  4:                      Blautia producta 1.055413e-08        TRUE      *
##  5:                   Blautia sp. N6H1-15 1.220307e-04        TRUE      *
##  6:               Clostridium perfringens 5.166682e-01       FALSE       
##  7:                 Enterocloster bolteae 1.313457e-09        TRUE      *
##  8:          Faecalibacterium prausnitzii 7.687366e-06        TRUE      *
##  9:               Faecalimonas umbilicata 1.039283e-07        TRUE      *
## 10:              Fusobacterium perfoetens 3.881664e-07        TRUE      *
## 11:                  Fusobacterium varium 2.208536e-06        TRUE      *
## 12:                Lactobacillus animalis 1.562315e-02        TRUE      *
## 13:               Lactobacillus johnsonii 5.637611e-01       FALSE       
## 14:                 Lactobacillus reuteri 1.498909e-01       FALSE       
## 15:                  Megamonas funiformis 7.241348e-05        TRUE      *
## 16:                 Megamonas hypermegale 2.581652e-08        TRUE      *
## 17:                 Megamonas rupellensis 7.436912e-07        TRUE      *
## 18:             Peptacetobacter hiranonis 6.127696e-13        TRUE      *
## 19:   Phascolarctobacterium succinatutens 6.797769e-06        TRUE      *
## 20:                  Phocaeicola plebeius 1.918687e-07        TRUE      *
## 21:                 Romboutsia timonensis 7.379722e-01       FALSE       
## 22:                 Streptococcus equinus 6.336182e-02       FALSE       
## 23:             Streptococcus lutetiensis 4.101400e-02        TRUE      *
## 24:                 Turicibacter sp. H121 1.922370e-01       FALSE       
## 25:                 [Ruminococcus] gnavus 9.990586e-01       FALSE       
##                                     taxon      p_value significant  label

PERMANOVA

Full-model PERMANOVA: source + dna.isolation.method

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Blocks:  strata 
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = otutab_t ~ sex + dna.isolation.methods + date, data = sampdat, method = distance, by = "terms", strata = sampdat$source)
##                       Df SumOfSqs      R2       F Pr(>F)    
## sex                    1   0.3730 0.02338  2.7969  0.001 ***
## dna.isolation.methods  1   3.2992 0.20679 24.7391  0.001 ***
## date                  13   3.7468 0.23485  2.1612  0.001 ***
## Residual              64   8.5350 0.53498                   
## Total                 79  15.9539 1.00000                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Summarised R² values Across Taxon Levels

Barplot

Lineplot

Summary of PERMANOVAs on Different Rank Levels

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